Method for inhibiting platelet aggregation

ABSTRACT

A method for inhibiting platelet aggregation in a patient in need thereof, comprising 1) administering to the patient a bolus injection of an active drug, in an amount of between about 25 μg/kg, and 2) administering to the patient, after the bolus injection, an intravenous infusion for a period of between about 12 hours and about 72 hours, of the active drug, in an amount of about 0.15 μg/kg/min, wherein the active drug is tirofiban or a salt thereof.

This application is a continuation of U.S. application Ser. No.10/427,436 (provisional application No. 60/380,084), filed May 6, 2002,which is incorporated in its entirety herein by reference.

BACKGROUND OF THE INVENTION

Platelet activation and aggregation are involved in unstable angina andacute myocardial infarction, in reocclusion following thrombolytictherapy and angioplasty, in transient ischemic attacks, and in a varietyof other vaso-occlusive disorders. When a blood vessel is damaged,either by acute intervention such as angioplasty, or, more chronically,by the pathophysiological processes of atherosclerosis, platelets areactivated to adhere to the disrupted surface and to each other. Thisactivation, adherence and aggregation may lead to occlusive thrombusformation in the lumen of the blood vessel.

Antiplatelet therapy has been used in a wide variety of cardiovasculardisease states and in conjunction with interventional therapy such ascoronary artery or peripheral bypass grafting, cardiac valvereplacement, and percutaneous transluminal coronary angioplasty.Inhibitors of the glycoprotein complex GP IIb/IIIa, including abciximab,tirofiban, and eptifibatide, are used intravenously to inhibit plateletaggregation. Platelet aggregation inhibition results in reducedincidences or reduced severity of adverse events such as death or damageto the heart. Typical use of these inhibitors involves initial bolusinjection and subsequent sustained infusion, for a period of hours ordays.

Holmes et al., Coronary Artery Disease (2001) 12:245-253, describe theefficacy of platelet aggregation inhibition induced by treatment withtirofiban hydrochloride in patients with unstable angina, non-ST-segmentelevation myocardial infarction or symtomatic coronary diseaseundergoing percutaneous coronary intervention. Patients received either0.4 μg/kg/min over 30 minutes followed by 0.1 μg/kg/min, or 10 μg/kgbolus injection followed by continuous infusion at 0.15 μg/kg/min.

Neumann, et al., J. Am. Coll. Cardiol. (2001) vol. 37, pp. 1323-1328,describe antiplatelet effects if tirofiban hydrochloride in patientsundergoing intracoronary stent placement for symptomatic coronary arterydisease. Patients received 10 μg/kg bolus injection followed bycontinuous infusion at 0.15 μg/kg/min for 72 hours.

Topol et al., N Engl J Med, (2001) vol. 344, pp. 1888-1894, describe theuse of tirofiban hydrochloride for the prevention of ischemic eventswith percutaneous coronary revascularization. Treated patients werethose scheduled to undergo a coronary stenting procedure of a newlystenotic or restenotic atherosclerotic lesion in a native vessel or abypass graft. Patients received 10 μg/kg bolus injection followed bycontinuous infusion at 0.15 μg/kg/min for 18-24 hours.

Kabbani et al. The American Journal of Cardiology (2002) vol. 89 pp.647-650, describe the use of tirofiban hydrochloride in patients havingan acute coronary syndrome in whom a percutaneous coronary interventionwas mandated. Patients received 10 μg/kg bolus injection followed bycontinuous infusion at 0.15 μg/kg/min for 18-24 hours. Results from thestudy show that the average inhibition of maximal aggregation during theperiod of time between 15 and 60 minutes following administration of thebolus dose was between 61% and 66%.

In each of the above studies, patients were treated either with no bolusdose of tirofiban hydrochloride or a 10 μg/kg bolus dose of tirofiban.The duration of continuous infusion was between 18 and 72 hours. In nocase was the bolus amount greater than 10 μg/kg.

We have now found that substantially more effective inhibition ofplatelet aggregation can be obtained by administering, to a patient inneed thereof, a bolus dose of tirofiban hydrochloride of about 25 μg/kg.The increased bolus dose greatly improves the overall plateletaggregation inhibitory effect of tirofiban therapy, providing anaggregation inhibition of greater than 90%, without the need to increasethe concentration of tirofiban hydrochloride solution delivered duringthe continuous infusion phase of tirofiban therapy, and without the needto extend the duration of the continuous infusion phase. Further, thisbenefit is achieved in the absence of increased undesirable sideeffects.

SUMMARY OF THE INVENTION

The invention is a method for inhibiting platelet aggregation in apatient in need thereof, comprising 1) administering to the patient abolus injection of an active drug, in an amount of about 25 μg/kg, and2) administering to the patient, after the bolus injection, anintravenous infusion of an active drug for a period of between about 12hours and about 72 hours, in an amount of about 0.15 μg/kg/min, whereinthe active drug is tirofiban or a salt thereof.

In a class of methods of the invention, the salt is tirofibanhydrochloride. In a subclass of the class, the amount of tirofibanhydrochloride in the bolus injection is 25 μg/kg. In another subclass ofthe class, the intravenous infusion is between 12 hours and 72 hours,e.g. between 18 hours and 72 hours.

The invention is also a method for reducing the risk of acute coronarysyndrome in a patient at risk to acute coronary syndrome, comprising 1)administering to the patient a bolus injection of an active drug, in anamount of between about 25 μg/kg, and 2) administering to the patient,after the bolus injection, an intravenous infusion for a period ofbetween about 12 hours and about 72 hours, of the active drug, in anamount of about 0.15 μg/kg/min, wherein the active drug is tirofiban ora salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

Tirofiban hydrochloride, commercially available as AGGRASTAT®, is anon-peptide antagonist for the glycoprotein IIb/IIIa fibrinogenreceptor. Tirofiban hydrochloride is chemically described asN-(butylsulfonyl)-O-[4-(4-piperidinyl)butyl]-L-tyrosinemonohydrochloride and structurally represented as

Tirofiban hydrochloride is also referred to as(2-S-(n-Butylsulfonylamino)-3[4-(piperidin-4-yl)butyloxyphenyl]propionicacid hydrochloride, and is described in U.S. Pat. No. 5,292,756.

Tirofiban hydrochloride and related pharmaceutically acceptable saltsare useful in the present invention. The term “pharmaceuticallyacceptable salts” means non-toxic salts of the compounds which include,but are not limited to, acetate, benzenesulfonate, benzoate,bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate,camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate,lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,oleate, oxalate, pamaote, palmitate, panthothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, tannate, tartrate, teoclate, tosylate,triethiodide, valerate.

Tirofiban, tirofiban hydrochloride, and other tirofiban salts, are alsocollectively referred to hereinafter as “active drug.”Pharmaceuticallyeffective amounts of the active drug are suitable for use in the methodsof the present invention. The term “pharmaceutically effective amount”means that amount of a drug or pharmaceutical agent that will elicit thebiological or medical response of a tissue, system or animal that isbeing sought by a researcher or clinician.

The methods of the present invention are useful in combination withother procedures for treating candidate patients, including proceduresinvolving treatments with other anticoagulants (e.g. heparin andwarfarin), thrombolytic agents (e.g. streptokinase and tissueplasminogen activator), and platelet antiaggregation agents (e.g.aspirin and dipyridamole).

The dosage regimen utilizing the active drug is selected in accordancewith weight of the patient; an ordinarily skilled physician orveterinarian can readily determine and prescribe the effective amount ofthe drug required to prevent, counter, or arrest the progress of thecondition in accordance with the present invention.

The active drug can be administered in admixture with suitablepharmaceutical diluents, excipients or carriers (collectively referredto herein as “carrier” materials) suitably selected with respect to theintended form of administration and consistent with conventionalpharmaceutical practices.

The methods according to the present invention for administering theactive drug are useful for treating patients where inhibition of humanor mammalian platelet aggregation or adhesion is desired. They areuseful in surgery on peripheral arteries (arterial grafts, carotidendaterectomy) and in cardiovascular surgery where manipulation ofarteries and organs, and/or the interaction of platelets with artificialsurfaces, leads to platelet aggregation and potential formation ofthrombi and thromboemboli. Methods of the invention may be used toprevent the formation of thrombi and thromboemboli. Other applicationsinclude prevention of platelet thrombosis, thromboembolism andreocclusion during and after thrombolytic therapy and prevention ofplatelet thrombosis, thromboembolism and reocclusion after angioplastyor coronary artery bypass procedures. The methods may also be used toprevent myocardial infarction.

The present invention is demonstrated in a study of patients with acutecoronary syndrome who are undergoing early coronary revascularizationwith percutaneous coronary angioplasty or atherectomy. Because ofunstable plaque with thrombus, percutaneous revascularization proceduresin these patients carry with them considerable higher morbidity thanprocedures performed in patients with stable coronary disease. Patientsare evaluated after treatment for acute coronary syndrome and mayrequire follow-up intervention associated with acute coronary syndrome,including coronary artery bypass grafting, repeat percutaneousintervention for acute ischemia, and insertion of a coronaryendovascular stent.

EXAMPLE 1

Treatment of Acute Coronary Syndrome

Eligible patients included those with an acute coronary syndrome in whoma percutaneous coronary intervention was clinically mandated. An acutecoronary syndrome was defined by the following criteria; ischemicsymptoms plus either 0.5 mm of ST segment depression on the ECG or anelevated troponin or creatine kinase MB fraction. Exclusion criteriaincluded treatment with an antiplatelet agent other than aspirin in theprevious 14 days, thrombolytic therapy within 24 hours, renalinsufficiency (creatinine greater than 2.5 mg/dl), and anycontraindication to treatment with a glycoprotein IIb-IIIa inhibitor.

Patients were treated with a 20 μg/kg bolus of tirofiban hydrochloridefollowed by a 0.15 μg/kg/min infusion for 18-24 hours or a 25 μg/kgbolus followed by the same infusion. Enrollment of subjects in the panelwith the 20 μg/kg bolus (n=15/panel) was completed and the clinicaleffects and pharmacodynamic properties were evaluated before subjectswere enrolled in the panel with the 25 μg/kg bolus. Patients weretreated with aspirin (325 mg before the procedure and daily) andunfractionated heparin (target activated clotting time 250 seconds).Clopidogrel (300 mg and then 75 mg daily)(commercially available asPLAVIX®) was administered at least 45 minutes after the start oftirofiban hydrochloride. All other medications were administered at thediscretion of the attending cardiologist. Sheath removal was performedwhen the activated clotting time was less than 175 seconds unless aclosure device was used.

Blood samples were obtained from a venous catheter for assessment ofplatelet function by light transmission aggregometry, rapid plateletfunction analyzer and flow cytometry before treatment and after 15, 30,45, and 60 minutes. Blood was obtained after 5 minutes for analysis byflow cytometry. Blood for light transmission aggregometry and rapidplatelet function analyzer was anticoagulated withD-Phe-Pro-Arg-chloromethyl ketone (38 μM) to avoid potentiallyconfounding effects of citrate on inhibitory properties of tirofibanhydrochloride (Rebello et al., J. Thromb. Thrombolysis (2000) vol. 9 pp.23-28). Blood for flow cytometry was anticoagulated with corn trypsininhibitor, a specific inhibitor of coagulation factor XIIa withouteffect on other coagulation factors (Schneider et al., Circulation(1997) vol. 96 pp. 2877-83). Aggregation (light transmissionaggregometry) of platelets was assessed in platelet rich plasma inresponse to 20 μM adenosine diphosphate (Chronolog). Maximal aggregation(ex vivo) after 4 minutes was determined. rapid platelet functionanalyzer was performed in accordance with manufacturer specificationswith thrombin receptor agonist peptide cartridges. Assessment of thecapacity of platelets to bind fibrinogen was performed as previouslydescribed (Holmes et al., Coron. Artery Dis. (2001) vol. 12 pp. 245-253;Kabbani et al., Circulation (2001) vol. 104 pp. 181-186). For flowcytometry, samples were processed and platelets were fixed at each site.

The study was designed to identify a bolus dose of tirofibanhydrochloride that inhibited platelet aggregation, on average, by atleast 90% with a lower 95% confidence interval of the extent ofinhibition of at least 85% at all times between 15 to 60 minutes afteronset of treatment. The occurrence of 3 major bleeding episodes (asdefined by the American College of Cardiology Task force (Cannon et al.J. Am. Coll. Cardiol. (2001) vol. 38 pp. 2114-30) in each panel was apre-specified criteria for termination of the study.

The activated clotting time at the time of percutaneous coronaryintervention was 249±24 seconds. No major bleeding episode occurred witheither the 20 μg/kg or 25 μg/kg bolus.

The average extent of inhibition of platelet aggregation assessed withlight transmission aggregometry (20 μM adenosine diphosphate) rangedfrom 84% to 89% from 15 through 60 minutes after the 20 μg/kg bolus andfrom 92% to 95% after the 25 μg/kg bolus of tirofiban (see Table 1).TABLE 1 % Inhibition of platelet aggregation 15-60 minutes after bolusinjection 10 μg/kg 20 μg/kg 25 μg/kg 61-66% 84-89% 92-95%

The antiplatelet effects of the 20 and 25 μg/kg bolus were evaluatedalso by flow cytometric determination of the capacity to bind fibrinogenin response to 1 μM adenosine diphosphate. The extent of inhibition wasgreater after onset of treatment with the high bolus dose.

Light transmission aggregometry and rapid platelet function analyzerwere performed with D-Phe-Pro-Arg-chloromethyl ketone as theanticoagulant and flow cytometry was performed with blood anticoagulatedwith corn trypsin inhibitor. Citrate and other chelators of calciumalter platelet reactivity and the inhibitory properties of GP IIb-IIIainhibitors (Rebello et al., J. Thromb. Thrombolysis (2000) vol 9 pp.23-28; Schneider et al. Circulation (1997) vol. 96 pp. 2877-83).Accordingly, the evlauations were performed with conditions that limitpotentially confounding influences of selected anticoagulants andsimulate intense exposure of platelets to multiple platelet agonistsduring thrombosis by using high concentrations of adenosine diphosphateand thrombin receptor agonist peptide.

The interval from 15 to 60 minutes after onset of treatment is acritical period during which iatrogenic vessel injury is induced and athrombogenic object (intra-coronary stent) is frequently introduced. Thepresent invention identifies a dosage range of tirofiban hydrochloridethat achieves an average inhibition of platelet aggregation of greaterthan 90% throughout the first hour after treatment. This dosage entailsan increase in the bolus amount as compared to conventional bolusamounts (from 10 to 25 μg/kg), but no change in the rate or duration ofthe infusion.

EXAMPLE 2

Intravenous Formulations

An intravenous dosage form of(2-S-(n-Butylsulfonylamino)-3[4-(piperidin-4-yl)butyloxyphenyl]propionicacid hydrochloride (Active I) is prepared as follows: Active I 0.5-10.0mg Sodium Citrate 5-50 mg Citric Acid 1-15 mg Sodium Chloride 1-8 mgWater for Injection (USP) q.s. to 1 L

Utilizing the above quantities, Active I is dissolved at roomtemperature in a previously prepared solution of sodium chloride, citricacid, and sodium citrate in Water for Injection (USP, see page 1636 ofUnited States Pharmacopeia/National Formulary for 1995, published byUnited States Pharmacopeial Convention, Inc., Rockville, Md., copyright1994).

EXAMPLE 3

Intravenous Formulations

A pharmaceutical composition was prepared at room temperature usingActive I, a citrate buffer, and sodium chloride, to obtain aconcentration of 0.25 mg/ml.

800 grams of water was introduced into a standard pharmaceutical mixingvessel. 0.25 grams of Active I was dissolved in the water. 2.7 gramssodium citrate and 0.16 grams citric acid were added to obtain afinished citrate concentration of 10 mM. 8 grams of sodium chloride wasadded. 200 grams of water was then added to achieve the desired finalconcentrations of ingredients. The resulting aqueous formulation had thefollowing concentrations: Ingredient Amount Active I 0.25 mg/ml citratebuffer 10 mM sodium chloride 8 mg/ml

The finished concentrated formulation is stored in a standard USP Type Iborosilicate glass container at 30-40 degrees C. Prior to compoundadministration, the concentrated formulation is diluted in a 4:1 ratioresulting in a finished concentration of 0.05 mg/ml and transferred toan infusion bag.

1. A method for reducing the risk of acute coronary syndrome in apatient at risk to acute coronary syndrome, comprising 1) administeringto the patient a bolus injection of tirofiban or salt thereof, inamounts sufficient to inhibit platelet aggregation of at least 85% atall times between 15 and 60 minutes after onset of treatment followingadministration of the bolus injection.
 2. The method of claim 1 whereinthe patient is undergoing early coronary revascularization withpercutaneous coronary angioplasty.
 3. The method of claim 1 wherein thepatient is undergoing early coronary revascularization with percutaneouscoronary atherectomy.
 4. The method of claim 1 wherein the acutecoronary syndrome requires insertion of a coronary endovascular stent.5. The method of claim 1 wherein acute coronary syndrome requires repeatpercutaneous intervention for acute ischemia.
 6. The method of claim 1wherein acute coronary syndrome requires coronary artery bypassgrafting.
 7. A method of preventing platelet thrombosis, thromboembolismand reocclusion during and after thrombolytic therapy in a patientcomprising 1) administering to the patient a bolus injection oftirofiban or salt thereof, in amounts sufficient to inhibit plateletaggregation of at least 85% at all times between 15 and 60 minutes afteronset of treatment following administration of the bolus injection.
 8. Amethod of preventing platelet thrombosis, thromboembolism andreocclusion after angioplasty or coronary artery bypass procedures in apatient comprising 1) administering to the patient a bolus injection oftirofiban or salt thereof, in amounts sufficient to inhibit plateletaggregation of at least 85% at all times between 15 and 60 minutes afteronset of treatment following administration of the bolus injection.
 9. Amethod of preventing myocardial infarction in a patient comprising 1)administering to the patient a bolus injection of tirofiban or saltthereof, in amounts sufficient to inhibit platelet aggregation of atleast 85% at all times between 15 and 60 minutes after onset oftreatment following administration of the bolus injection.
 10. A methodof administering to a patient a bolus injection of tirofiban or saltthereof, in amounts sufficient to inhibit platelet aggregation of atleast 85% at all times between 15 and 60 minutes after onset oftreatment following administration of the bolus injection.
 11. Themethod of claim 10 wherein the platelet aggregation occurs in peripheralarteries.